Fluorescent dyes are relied upon in a wide variety of fields, particularly in vitro and in vivo fluorescence microscopy, such as used in wide-field, scanning confocal, and Total Internal Reflection Fluorescence Microscopy (TIRF) used for whole cell and single-molecule imaging. The use of fluorescent labels with antibodies, DNA probes, biochemical analogs, lipids, drugs, cells and polymers has expanded rapidly in recent years. High-quantum yield, stable fluorescent species are generally preferred in fluorescence microscopy.
Of the dyes commonly used in bioanalytical studies, the cyanine dyes (e.g., Cy3, Cy5, and Cy7) are particularly well known. The cyanine dyes have proven useful in a wide range of applications, including the labeling of a variety of materials (e.g., hydrophilic and hydrophobic surfaces of various materials, including nanoparticles), in microscopic studies of living cells, and in single-molecule imaging, due in large part to their large extinction coefficients (ca. 250,000 M−1 cm−1 for Cy5) and quantum yield (approximately 0.3 for Cy5). The dyes are also widely used as fluorescent probes in DNA sequencing, cellular analysis (e.g. molecular beacons and single-particle tracking), flow cytometry, and super-resolution imaging.
However, the utility of these dyes is substantially hindered by undesirable photophysical properties that lead to transient and/or permanent dark states. It is believed that these dark states arise via electronic transitions from the singlet ground and/or excited states to triplet dark states. From triplet states, deleterious physical modifications or damage can occur to the dye. In particular, such processes tend to limit photon emission from the fluorophore and often result in stochastic “blinking” events and irreversible photobleaching. Blinking and photobleaching phenomena occur in all fluorescence applications but are particularly pronounced in experiments demanding intense illumination, including confocal imaging of cells and single-molecule fluorescence methods.
It has recently been discovered that certain small organic molecules can favorably affect the intensity and photostability of Cy5 when included in solution during imaging experiments (Rasnik et al. Nature Methods 2006; Aitken et al. Biophys J. 2008; Dave et al. Biophys. J 2009). These compounds are generally referred to as triplet state quenchers (TSQs) as they are thought to operate by reducing the lifetime of triplet dark states that occur with finite probability as a consequence of fluorophore excitation. Some examples of TSQs include Trolox, p-nitrobenzyl alcohol (NBA), β-mercaptoethanol (BME), mercaptoethylamine (MEA), n-propyl gallate, 1,4-diazabicyclo[2.2.2]octane (DABCO), and cyclooctatetraene (COT). As photobleaching is thought to principally occur from triplet excited states, TSQs, by reducing excursions to triplet states, have the propensity to: 1) increase the mean intensity of stochastically emitting fluorophores; 2) reduce the variance in photo-emission rate and 3) reduce the probability of photobleaching, thereby extending the duration of time over which photons are emitted.
Despite the potential benefits of their use, TSQs generally have been significantly limited in their use for in vitro and cell-based imaging experiments. At least one significant limitation in the experimental implementation of using TSQs in solution is due to their relatively poor aqueous solubility (generally <2 mM), their varied solubilities in aqueous buffers with distinct ionic strengths, and their potential to disrupt lipid bilayers and biological molecules which can render them toxic to cells and potentially disruptive to the biological activities under investigation. Moreover, the existing methodologies do not permit specific and tailored distances to be maintained between the fluorophore and TSQ, nor do they permit specific binding of a fluorophore-TSQ pair, separated by a specified distance, to a biomolecule or other molecule or material of interest. The ability to select and tailor these distances and binding locations would provide fluorophores that are selectively adjusted in their photophysical properties, which could be modified or optimized to meet the demands of their intended use and the localized molecular environment.